Cardiovascular disease remains the leading cause of death in North America and Western Europe. The majority of these deaths are due to myocardial or cerebral infarction, with atherosclerosis being the principal cause (McGill Jr., 1988, Clin Chem 34: 1333–1339). It has long been recognized that cholesterol plays a role in the formation of atherosclerotic plaques; the compound was first noted as a lesion component almost a century ago by Windaus. More recently, it has become clear that total serum cholesterol consists of subfractions that must be considered individually in order to assess the risk profile. In particular, above normal levels of cholesterol sequestered in high density lipoproteins (HDL cholesterol, or HDL-C) carries a message opposite to that implied by elevated low density lipoprotein cholesterol (LDL-C). The principal physiochemical risk factor is a high serum level of the primarily atherogenic LDL cholesterol (Schwartz et al., 1991, Clin Cardiol 14: 11–116), since these lipoproteins promote the deposition of plasma lipids in the artery wall and elicit the formation of fatty streaks and/or atherosclerotic plaques. LDL-C is now recognized as a key factor in the development of atherosclerosis. On the other hand, HDL is associated with decreased incidence of atherosclerosis. Produced mainly in the liver and intestine (it can also be derived as metabolic by-products of chylomicron and VLDL catabolism), the main function of HDL is the transport of cholesterol from the peripheral cells to the liver, i.e., “reverse cholesterol transport” (Golmset, 1968, J Lipid Res 9: 155–167).
While a wide variety of methods have been proposed for the determination of serum LDL cholesterol, including electrophoresis, HPLC, sequential and density gradient ultracentrifugation, precipitation-based methods, and immunoseparation, the standard routine laboratory test is an indirect one. The Friedewald formula has proven to be extraordinarily useful in providing a close approximation to true LDL values, particularly since no practical alternative has emerged for large scale routine testing. Despite a good correlation between measured LDL cholesterol levels and those calculated using the Friedewald formula, some limitations are inevitably encountered. For example, the formula cannot be employed for non-fasting samples, and is inaccurate for samples with hypertriglyceridemia (serum triglyceride levels exceeding 4.5 mmol/L)—a common finding in uremic patients—or for patients with dysbetalipoproteinemia (Nauck et al., 1996, Clin Nephrol 46: 319–325). Furthermore, this method needs measurement of three parameters to obtain LDL-C and therefore a higher variability is unavoidable. The intraclass correlation coefficients demonstrate a poor concordance between calculated and measured LDL cholesterol, both in patients and controls (Senti et al., 1996, Angiology 47: 241–246). Given the limitations of current methods and the high prevalence of CHD in North America, there is a great demand for an accurate and preferably automated method for the determination of LDL-C. Here, we propose a method based upon infrared (IR) spectroscopy for the simultaneous determination of both HDL and LDL cholesterol.
IR spectroscopy, a technique that utilizes infrared light absorption patterns to obtain structural and analytical information, has been applied previously in studies of lipoprotein structures. For instance, Scanu et al first employed IR spectroscopy to examine the thermal behavior of apoB (Scanu et al., 1969, PNAS 62: 171–178). Later on, IR was used to elucidate the secondary structure of apoIB, first qualitatively using resolution-enhancement techniques (Herzyk et al., 1987, Biochim Biophys Acta 922: 145–154) and then quantitatively using curve fitting of deconvolved spectra (Goormaghtigh et al., 1989, Biochim Biophys Acta 1006: 147–150). More recently, Goormaghtigh et al have utilized IR spectroscopy to reveal the structure of the lipid attached proteins that remain following proteolytic digestion of solvent-exposed regions (Goormaghtigh et al., 1993, Biochemistry 32: 6104–6110).
Previous studies from our group have demonstrated that a wide array of serum (Shaw et al., 1998, Ann Clin Biochem 35: 624–632) and urine (Shaw et al., 2000) analytes may be determined via IR spectroscopy of films dried from the fluid of interest. The present study reveals that the spectra of HDL and LDL cholesterol complexes are distinctive enough to permit the separate quantitation of HDL cholesterol and LDL cholesterol based upon the IR spectra of dried serum films. This finding offers a simple, reagent-free method for the simultaneous determination of HDL cholesterol, LDL cholesterol, and, as previously demonstrated (Shaw et al., 2000), total cholesterol and triglycerides.
U.S. Pat. No. 5,856,196 teaches a method of determining the level of phospholipids such as dipalmitoyl phosphatidyl choline in amniotic fluid which requires that the polar head group from the phosphoglycerides in the sample. As can be seen, this requires an added step wherein the sample must be treated prior to analysis.
U.S. Pat. No. 5,424,545 teaches a method for measuring the blood concentration of analytes such as glucose which relies on colorimetry rather than spectrophotometric means.
U.S. Pat. No. 6,026,314 teaches a noninvasive method for measuring blood component concentrations, for example, glucose or cholesterol, which comprises irradiating infrared light onto the skin of a subject.
As can be seen, the previous two patents claim methods to estimate concentrations of blood analytes such as cholesterol. However, as discussed above, cholesterol level alone is not a suitable predictor for cardiovascular health. That is, the prior art does not provide a definitive method for measuring LDL-cholesterol.
In the present study, we combined IR spectroscopy with a powerful quantitation algorithm, namely partial least squares (PLS), to assess the potential application of IR spectroscopy for the simultaneous determinations of several cardiovascular risk markers, as described below.